Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress Oxidation of PUFAs in membrane phospholipids leads to the formation of molecular fragments, some of which are diffusible throughout the cell, and others that remain phospholipid-bound. Many of the oxidation products of both classes are reactive electrophiles. Past research has demonstrated that diffusible electrophilic products of PUFA oxidation such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) form adducts with proteins and DMA and lead to cell cycle arrest and/or apoptosis. Our laboratory has carried out extensive studies of the mechanism of HNE-induced apoptosis, which has allowed us to identify the specific apoptotic pathways involved, and to assess the global transcriptional response to HNE-induced damage. Despite this progress in defining the role of diffusible electrophiles in the pathophysiology of oxidative stress, relatively little is known about the contribution of phospholipid-bound electrophiles. Such species differ from diffusible electrophiles with regard to susceptibility to intracellular detoxification and accessibility to target proteins. We hypothesize that electrophilic derivatives of phospholipids are important effectors of oxidative damage and that they differ from structurally related, diffusible electrophiles in their reactions with proteins and the cellular responses they elicit. We will test this hypothesis by determining the cytotoxicity of phospholipid electrophiles generated in Project 1 in a high throughput screen. The mechanism of toxicity will be determined for active compounds, and for those found to induce apoptosis, we will use microarray analysis to determine the global transcriptional changes evoked by these compounds. We will confirm the results using RT-PCR, Western blotting, and transcriptional activation assays. In collaboration with Project 4, we will synthesize phospholipid and diffusible electrophiles capable of being affinity tagged, and use them to determine the protein targets of both classes of compounds. These collective data will be used to identify the major transcriptional pathways activated in response to electrophile exposure. We will examine the role of these pathways in order to determine how they act to elicit cytoprotective or proapoptotic responses in the cells. The data derived from all of these studies will be used in conjunction with our previous data on diffusible electrophiles to determine the relative toxicity of and to determine differences in cellular responses to the two classes of oxidation products. These findings will lead to a better understanding of the pathophysiology of oxidative stress and provide the foundation for a rational approach to the development of better therapeutic modalities for diseases in which oxidative stress plays a major role.